My CAP and PolyA RNA has been quantified on the nanodrop and all the samples show high concentration as well as the ration between 260/280. A is over 3 in all of them.
Does that mean I have extra material in my RNA sample and how do I clean it?
I've performed TurboDNase treatment and precipitated the probe after capping and poly-tailing it.
Thank you!
I am not an expert in RNA capping and tailing but I guess that your RNA are enriched in adenine. I know that some commercial kit allow to tail RNA with at least 150 adenine ... and adenine 260/280 ratio is 4.5 ... so maybe your 260/280 > 3 is because of adenine enrichment
Hi there,
What is over 3? Absorbance at 260nm? Absorbance at 280nm? Both? What about the value of 260/280 ratio? If the latter is above 2, it means either the baseline hasn't been properly done and/or there is something in your prep contributing preferentially at 260nm (phenol and guanidine derivatives are often observed in nucleic acid preps). For your info:
http://www.nanodrop.com/Library/T123-NanoDrop-Lite-Interpretation-of-Nucleic-Acid-260-280-Ratios.pdf
Abnormally high 260/280 might indicate degraded RNA. I would check aliquots of your RNA samples on a (preferably denaturing) agarose gel and check for the presence and intensity of the rRNA bands.
Best regards, Christian
Thank you for all the answers! David, I was thinking the exact same thing. I think if I run it on a gel, it should let me know my RNA is intact? Christian, could you send me a protocol for the gel?
Thank you!!
Hello Monika,
in the attachment you will find the protocol for preparing denaturing agarose gels for RNA analysis. I recommend to wear gloves when you prepare these gels (to prevent RNase contamination and as protection against toxic chemicals).
Regards, Christian
Hi Monika,
I agree with checking the RNA integrity of your sample. Check out whether there's a bioanalyzer in your institute, that would save you a lot of time and effort making tricky gels. Make sure you know what sort of bands you're expecting from your sample, if you've depleted from rRNA you might not see much as this is the main component of your totalRNA.
If you later confirm that your RNA is degraded, I would try to eliminate the DNAse treatment. In my experience, DNAse treatment always degrades, if partially, the RNA in your sample.
Good luck!
Anna
Can you please tell me whether the RNA isolated is of good quality or not (Photo attached) ?
The quality of the electrophoretic separation is problematic. It is not possible to say whether your RNA isolation was successful or not. How did you prepare your samples and the gel?
Regards,
Christian
Hi Christian,
-By using nanodrop, i am getting conc range from 100ng/ul to 250ng/ul.
- Agarose gel electrophoresis was used for quality check.
- cDNA was prepared and was confirmed by GAPDH primer and got good results.
Can I consider this RNA for real-time PCR.
Hi Lokesh,
it would be better to use a denaturating agarose gel. Usually the intensity and the ratio of certain rRNA bands gives a good clue whether your RNA is degraded or mostly intact. On the other hand, you could use your cDNA to test whether you can ampify a housekeeping gene (e. g., actin, tubulin, etc.). It is important that the amplification product is not too small, though. Very important is to rule out the presence of contaminating genomic DNA. Don't hesitate to ask if you would like to know more.
Regards, Christian
Thank You so much @Christian :)
Now my question is as I already mentioned above that I prepared cDNA and then checked by using GAPDH on simple PCR and got amplified product of same size (200bp).
So can I use it for RT-PCR???
In this case, I would proceed with the RT-PCR experiments. Good luck!
Cheers,
Christian
I had done RTPCR for my samples using Sybergreen . Can you guys please tell me whether my Ct values are ok or not?(File attached)
Thanks in advance
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